1. Field
The disclosed subject matter relates to inhibitors of tyrosine kinase receptors (RTKs) such as EGFR, and methods of screening for compounds which inhibit RTK activity. In particular, the disclosed subject matter relates in inhibitors of RTK dimerization including EGFR dimerization, and methods of screening for such inhibitors.
2. Introduction
Protein kinases (PKs) are enzymes that catalyze the phosphorylation of hydroxy groups on tyrosine, serine and threonine residues of proteins. The consequences of this seemingly simple action are profound; cell growth, differentiation and proliferation; i.e., virtually all aspects of cell life, in one way or another, depend on PK activity. Abnormal PK activity has been related to a host of disorders, ranging from relatively non life-threatening diseases such as psoriasis to extremely virulent diseases such as glioblastoma (brain cancer).
Certain growth factor receptors exhibiting PK activity are known as receptor tyrosine kinases (RTKs). RTKs comprise a large family of transmembrane receptors with diverse biological activity. As present, at least nineteen (19) distinct subfamilies of RTKs have been identified. One RTK subfamily contains the insulin receptor (IR), insulin-like growth factor I receptor (IGF-IR) and insulin receptor related receptor (IRR). IR and IGF-IR interact with insulin to activate a hetero-tetramer composed of two entirely extracellular glycosylated alpha subunits and two beta subunits which cross the cell membrane and which contain the intracellular tyrosine kinase domain. The Insulin-like Growth Factor-1 Receptor (IGF-1R), and its ligands, IGF-1 and IGF-2, are abnormally expressed in numerous tumors, including, but not limited to, breast, prostate, thyroid, lung, hepatoma, colon, brain and neuroendocrine. A more complete listing of the known RTK subfamilies is described in Plowman et al., KN&P, 1994, 7(6):334-339 which is incorporated by reference in its entirety herein.
RTKs have been implicated in a host of pathogenic conditions including cancer. Other pathogenic conditions, which have been associated with abnormal RTK activity include, without limitation, psoriasis, hepatic cirrhosis, diabetes, atherosclerosis, angiogenesis, restenosis, ocular diseases, rheumatoid arthritis and other inflammatory disorders, autoimmune diseases and a variety of renal disorders.
The epidermal growth factor receptor (EGFR) is a prototypical RTK. The EGF receptor stimulates a complex signaling cascade that ultimately promotes cell proliferation, survival, and migration (Yarden, Y. & Schlessinger, J., Biochemistry 26, 1443-1451, 1987). Not surprisingly, perturbation of the EGF receptor system leads to a variety of tumors in organs including breast, brain, lung, ovary, and prostate (Normanno, N., et al., Gene 366, 2-16, 2006). In particular, 70-80% of metaplastic breast carcinomas over-express the EGF receptor (Reis-Filho, J., et al., Breast Cancer Research 7, R1028-R1035; http://breast-cancer-research.com/content/7/6/R10282005), and 40-80% of non-small cell lung cancers arise from EGF receptor over-expression and/or mutation (Grandis, J. R. & Sok, J. C., Pharmacol. Ther. 102, 37-46, 2004).
As a validated target in oncology, EGFR has been extensively studied and its intracellular signaling pathways mapped as described in Oda et al. Mol Systems Biol, 2005, 1(1): 1-17 which is incorporated by reference in its entirety herein. EGFR and ErbB2 belong to the ErbB family of receptor tyrosine kinases (RTK). They are characterized by an extracellular ligand-binding domain, a single transmembrane helix, an intracellular kinase domain, and a C-terminal tail (FIG. 1A). The receptors are thought to exist predominantly as monomers in the plasma membrane. Upon binding of ligands to the extracellular domain, EGF and ErbB receptors homo- or heterodimerize with each other (FIG. 1B). Receptor dimerization leads to kinase activation, resulting in transphosphorylation of specific tyrosine residues within the C-terminal tail of the extracellular domain of the receptor which facilitate dimerization. In particular, single mutation to either Y246 or Y251, two strictly conserved tyrosines on the “arm”, is enough to completely abolish dimerization (Dawson, J. P., et al., Mol. Cell. Biol. 25, 7734-7742, 2005; Walker, F., et al., J. Biol. Chem. 279, 22387-22398, 2004).
ErbB receptor dimerization is a pre-requisite for receptor activation and is driven by interactions between the extracellular domains (ECD) of the two partners. In the inactive monomeric state, the EGFR ECD adopts a tethered conformation where a long loop from domain II, known as the dimerization arm, is held between domain II and domain IV. Binding of growth factors like EGF to the ECD induces a large conformational arrangement (FIG. 2) where the dimerization arm is exposed to the environment in a conformation known as the extended conformation.
EGFR or ECDs thereof have been crystallized either with or without a ligand bound, and three-dimensional structures, including atomic coordinates, have been reported. (Ogino, H., et al., Cell 110, 775-787, 2002; Cho, H.-S., et al., Science 297, 1330-1333, 2002; Garrett, T. P. J. et al., Cell 110, 763-773, 2002). Atomic coordinates are available on the internet at http://www.rcsb.org/pdb/home/home.do (Berman, H. M., et al., Nucleic Acids Research 28, 235-242, 2000). The crystal structures show that the aromatic rings of Y246 and Y251 on one monomer pack nicely into a pair of adjacent “pockets” on the other monomer (FIG. 1). The crystal structures of the EGFR extracellular domain homodimer show that the most extensive part of the dimer interface is centered on the intermolecular interaction between the dimerization arms of two monomers. The dimerization arm (residues 242-259) buries more than 800 Å2 of surface area and is specific to the ErbB family receptors. EGFR with mutations on the dimerization arm fail to form dimers confirming the critical role of the “arm” in dimerization. In particular, single mutation to either Y246 or Y251, two strictly conserved tyrosines on the “arm”, is enough to completely abolish dimerization. The crystal structure shows that the aromatic rings of Y246 and Y251 on one monomer pack nicely into a pair of adjacent “pockets” on the other monomer (FIG. 2-3). In addition to the van der Waal interactions, the hydroxyl group of Y246 forms hydrogen bonds with G264 and C283, and has been suggested to be critical since dimerization is abolished in a Y246F mutant.
Given its strong association with cancer, the EGF receptor is a validated target in the emerging paradigm of mechanism-based cancer therapeutics (Ciardiello, F. Future Oncology 1, 221-234, 2005; Hynes, N. E. & Lane, H. A., Nat. Rev. Cancer 5, 341-354, 2005; Marshall, J., Cancer 107, 1207-1218, 2006). Current EGF receptor-directed strategies include monoclonal antibodies that target the extracellular domain (Kirkpatrick, P., et al., Nat. Rev. Drug Discov., 3, 549-550, 2004; Saltz, L., et al., Nat. Rev. Drug Discov. 5, 987-988, 2006), and small-molecule tyrosine kinase inhibitors that compete with ATP at the nucleotide binding site of the kinase domain (Dowell, J., et al., Nat. Rev. Drug Discov. 4, 13-14, 2005; Moy, B., et al., Nat. Rev. Drug Discov. 6, 431-432, 2007; Muhsin, M., et al., Nat. Rev. Drug Discov. 2, 515-516, 2003). These drugs have utility, but show highly variable efficacy in clinical applications (Pao, W., et al., PLoS Medicine 2, e73 2005; Perez-Soler, R., et al., Oncologist 9, 58-67, 2004). Recent reports showed that some of the clinical variability is due to an increasing number of cases where tumors develop resistance to the tyrosine kinase inhibitors by mutation of residues within the ATP binding site of the kinase domain (Bell, D. W., et al., Nat. Genet. 37, 1315-1316, 2005; Sharma, S. V., et al., Nat. Rev. Cancer 7, 169-181, 2007).